We have continued our studies on the metabolite of thotropin releasing hormone known as histidyl-proline diketopiperazine. During the past year, we have developed an antiserum in rabbits that is highly specific for histidyl-proline diketopiperazine. This antiserum does not bind thyrotropin releasing hormone or other related peptides and can distinguish between the natural form of histidyl-proline diketopiperazine and a diastereoisomer. The antiserum was used as a tool for developing an isolation and assay procedure for histidyl-proline diketopiperazine from rat brain. It was found that the cyclic dipeptide occurs naturally in a bound form associated with some material of molecular weight approximately 70,000. A procedure was devised for releasing the low molecular weight peptide from the bound form. The concentration of histidyl-proline in rat brain was found to be approximately 2.5 times higher than that of thyrotropin releasing hormone. A regional distribution of the peptide indicated that it was found in highest concentrations in pituitary and hypothalamus.